Low-temperature phenol-degrading microbial agent: construction and mechanism

In this study, three cold-tolerant phenol-degrading strains, Pseudomonas veronii Ju-A1 (Ju-A1), Leifsonia naganoensis Ju-A4 (Ju-A4), and Rhodococcus qingshengii Ju-A6 (Ju-A6), were isolated. All three strains can produce cis, cis-muconic acid by ortho-cleavage of catechol at 12 ℃. Response surface methodology (RSM) was used to optimize the proportional composition of low-temperature phenol-degrading microbiota. Degradation of phenol below 160 mg L−1 by low-temperature phenol-degrading microbiota followed first-order degradation kinetics. When the phenol concentration was greater than 200 mg L−1, the overall degradation trend was in accordance with the modified Gompertz model. The experiments showed that the microbial agent (three strains of low-temperature phenol-degrading bacteria were fermented separately and constructed in the optimal ratio) could completely degrade 200 mg L−1 phenol within 36 h. The above construction method is more advantageous in bio-enhanced treatment of actual wastewater. Through the construction of microbial agents to enhance the degradation effect of phenol, it provides a feasible scheme for the biodegradation of phenol wastewater at low temperature and shows good application potential.


Introduction
As an important organic chemical raw material, phenol is widely used in organic material synthesis, pharmaceuticals, and many other fields. Industrial wastewater from oil refineries, coking coal manufacturing, coal processing plants, and Communicated by Arivalagan Pugazhendhi.

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193 Page 2 of 14 paper mills usually contains a variety of organic compounds including phenol (Rea et al. 2023;Singh et al. 2022;Sun et al. 2023). Due to the strong bio-toxic and carcinogenic properties of phenol-containing wastewater (Zhou et al. 2019), the discharge of unregulated phenol-containing wastewater poses a great threat to the ecological environment and human health (Sachan 2019). Phenol poses a toxic threat to aquatic microorganisms when the concentration of phenol in water exceeds 0.05 mM (Dos Santos et al. 2009).
Research on microbial degradation of phenol has been further enhanced in recent years due to the inexpensive, efficient, and environmentally friendly properties of biological methods (Hou et al. 2021;Su et al. 2019). It has been reported that a variety of microorganisms can metabolize and mineralize phenol under medium-temperature aerobic conditions, such as Pseudomonas sp. (Kumar et al. 2005), Rhizobia (Lefebvre and Moletta 2006), Acinetobacter (Shahryari et al. 2018), Rhodococcus sp. (Paisio et al. 2012;Wen et al. 2020), etc. The biodegradation process is controlled by enzymes (Dalvi et al. 2012;Nandy et al. 2021). The intermediates produced by phenol biodegradation are catalyzed by the corresponding enzymes to generate pyruvate, acetic acid, succinic acid, and acetyl CoA (Loh and Chua 2002). Catechol is the most common intermediate product of phenol biodegradation. The degradation of catechol is usually via the ortho-or meta-cleavage pathway. The meta-cleavage pathway of catechol is commonly observed in mesothermal and thermophilic Bacilli (Ali et al. 1998). Vogtd et al. (2004) detected catechol 1,2-dioxygenase activity during the degradation of chlorobenzene at 14 ℃ using Pseudomonas veronii B547. Bai et al. (2022) found that Rhodococcus qingshengii FF could convert catechol to cis, cis-muconic acid at room temperature. Ortho-cleavage is considered a more efficient degradation route because it consumes less energy.
Currently, the biodegradation of phenol under pure culture conditions is relatively well studied. However, the degradation of phenol is a multifactorial process in which there is a complex influence between external environmental factors and the microorganism's own metabolism. For example, microorganisms are not conducive to organic matter degradation at low temperatures and in the presence of complex pollutants, and most microorganisms will enter a viable but non-culturable state (VBNC) (Wang et al. 2020), in which although microorganisms can survive, their own metabolism rate is slow, so it is difficult to achieve the desired effect in practical applications. It is particularly important to construct a low-temperature microbial agent to cope with the degradation of phenol under unfavorable conditions. In this study, three strains of low-temperature phenol-degrading strains were screened by enrichment culture, and their degradation characteristics and degradation pathways for phenol degradation process were investigated. The results of response surface experiments on the factors influencing phenol biodegradation were also used to construct the lowtemperature phenol-degrading microbiota. The phenol degradation kinetics of the microbiota were analyzed. The three strains were fermented separately and mixed according to the results of response surface methodology to construct microbial agent. The results showed the good potential for engineering applications of low-temperature microbial agent.

Culture enrichment, isolation, and batch experiments
The strains were obtained from the activated sludge of a petrochemical wastewater plant in winter in Xinjiang, China, and the enrichment culture was performed by artificial gradient water distribution (Girolamini et al. 2022;Collado et al. 2013). After 66 days of enrichment culture, the culture was serially diluted with sterile phosphate buffer solution (50 mM PBS, pH 7) and evenly spread on MSM medium containing 100 mg L −1 phenol, 2% agar with a smear stick. The culture was incubated at a constant temperature of 12 °C for 14 days, and six colonies growing on the plates were selected for further purification and isolation. The process was repeated three times and finally three strains were isolated from the sludge that were able to completely degrade phenol under low temperature.
Unless otherwise specified, all batch experiments were carried out in 250 mL conical flask under aerobic conditions. Phenol was biodegraded in 100 mL MSM medium at OD 660 0.3 ± 0.02, pH 7.0 ± 0.1, using an appropriate amount of phenol as the substrate. The medium was incubated in a constant temperature shaker at 12 °C and 150 rpm, and the residual phenol concentration in the medium was measured by periodic sampling.

Response surface experiments on the proportion of low-temperature phenol-degrading microbiota
The degradation experiment was carried out in MSM medium (pH 7.0) containing 100 mg L −1 phenol. Control the initial OD value to 0.3 ± 0.02, culture in a shaker at 12 ℃ and 150 rpm for 24 h, and determine the residual phenol content in the system according to the method in "Analysis methods". The central composite surface center (CCFD) design of the design expert 10 software was used to optimize phenol removal by selecting three independent variables (the proportion of the three strains added) (Varmira et al. 2016). The ranges and levels of the variables (high and low) studied in the experiment are given in Table 1.

Preparation of crude enzyme extracts
Ultrasonic crushing method was used to crush the above collected bacteria, the total working time was 3 min. The ultrasonic working procedure was 30-s interval after every 30 s of ultrasound, all the ultrasonic work was performed in ice bath (Margesin et al. 2004). Then, the lysing solution was centrifuged at 4 ℃ for 20 min at 12,000 rpm and the supernatant was collected, which was the cell-free crude enzyme extracts.

Enzymatic assays
The protein concentration in crude enzyme solution was determined by the Bradford method (Bradford 1976). The method of PheA assay mainly consisted of a reaction substrate consisting of 50 mM Tris-HCl buffer (pH 8.0) containing 2.5 mM phenol and 1 mol L −1 NADH. 1 mL of reaction substrate was added to 100 μL of enzyme extract and incubated at 30 °C for 30 min at constant temperature. The initial and final absorbance was measured at 340 nm using a UV-vis spectrophotometer (JASCO, UV-560, Japan).
The catechol 1,2-dioxygenase activity assay (C12O) and catechol 2,3-dioxygenase activity assay (C23O) were performed with reference to the methods in the literature (Mahiudddin et al. 2012;Nakazawa and Nakazawa 1970), absorbance was read at 260 nm and 375 nm, respectively. The specific activity is the number of units of enzyme activity per mg of cellular protein.

Extraction and derivatization of phenol intermediates
The analysis of phenol degradation intermediates of the three strains was performed in a system with a phenol concentration of 200 mg L −1 . The supernatant was collected after an interval of 24 h and centrifuged at 10,000 rpm for 20 min at 4 ℃. The supernatant was adjusted to pH 2 with 6 mol L −1 HCl, extracted three times with 10 mL ethyl acetate, combined with the organic phase after three extractions, and dehydrated with excess anhydrous sodium sulfate. The resulting sample was concentrated by rotary evaporation to approximately 0.5 ml, and 200 µL of N, O-bis(trimethylsilyl)acetamide (Nie et al. 2015) was added to the resulting concentrated intermediate, fixed to 1.0 mL with ethyl acetate, and then derivatized in a water bath at 60 °C for 30 min. Afterwards, the samples were measured by GC-MS in a brown GC vial through a glass syringe with a 0.22 μm organic filter membrane. All instruments in direct contact with the samples during the experimental operation were glass instruments and were used after 2 h of heating at 400 °C.

Fermentation culture
The fermentation medium was composed of glucose 5 g L −1 , yeast extract 5 g L −1 and MSM medium without ammonium sulfate for high-density fermentation of pure and mixed microbiota, respectively. The fermentation conditions were aerobic fermentation at 30 ℃, aeration of 100 mL min −1 , stirring speed of 60 rpm, inoculation of fermentation seeds at 5% by volume (v/v) (incubated by LB liquid medium to OD 660 value of 1) and fermentation for 48 h. The lowtemperature phenol-degrading microbial agent was prepared in two ways: one was to mix the monocultures mono cultures separately into the fermentation medium for 48 h and then mix them according to the optimal ratio of microbiota obtained from the results of RSM experiments (fermentation first and then mixing). The other was to ferment the microbiota into the fermentation medium at a constant temperature according to the optimal ratio (mixing first and then fermentation). The degradation medium was MSM medium containing 200 mg L −1 phenol.

Analysis methods
Cell concentration in the samples was determined by measuring the optical density at 660 nm by UV-vis spectrophotometer. Samples were taken periodically and the supernatant after centrifugation at 1000 rpm for 5 min was passed through a 0.22 μm filter membrane (polyether sulfone PES). The phenol detection was performed with methanol (70%): water (30%) as the mobile phase at a flow rate of 1 mL min −1 using HPLC (High-Performance Liquid Chromatography) (Shimadzu, LC-20AT, Japan) equipped with a UV diode array detector and a C18 reversed-phase column (particle size 5 μm, length 250 mm, tube diameter 4.6 mm). The concentration of phenol in the system was analyzed at a sample volume of 10 μL, column chamber temperature of 40 °C and wavelength of 271 nm.

Screening of low-temperature phenol-degrading strains
Sludge from a petrochemical wastewater treatment plant in Xinjiang, China, was domesticated for 66 days [ Fig. 1(a)]. Three strains were screened out, which can use phenol as the sole carbon source at 12 ℃ and can completely mineralize and degrade phenol. The results of 16 s rRNA gene sequencing showed that the three strains were strains Pseudomonas veronii Ju-A1 (Ju-A1), Leifsonia naganoensis Ju-A4 (Ju-A4), Rhodococcus qingshengii Ju-A6 (Ju-A6) (See Supplementary Material). The degradation of phenol at different concentrations by three strains was investigated at low temperature [ Fig. 1(b)]. At the degradation of 100 mg L −1 phenol, the degradation rate of Ju-A4 was 100% in 20 h, with a maximum degradation rate of 8.53 mg h −1 . The degradation rates of Ju-A1 and Ju-A6 were 48.5% and 42.4% at 20 h. The average and maximum degradation rates of Ju-A4 were decreased by 14.75% and 10.08%, respectively, when the concentration was increased to 200 mg L −1 phenol. The average degradation rate of Ju-A1 was 8.1% higher than that of Ju-A6, but the average degradation rates of both strains were lower than that of Ju-A4.

Determination of key enzyme activity of phenol-degrading strains at low temperature and analysis of intermediates
The activities of phenol hydroxylase (PheA), catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in the crude enzyme solution of three low-temperature phenol-degrading strains were determined. Phenol hydroxylase, also known as phenol 2-monooxygenase (EC 1.14.13.7). It is a NADPH-dependent flavoprotein monooxygenase which can add a single oxygen atom to benzene ring (Singh et al. 2019). This reaction is the limiting step of phenol biodegradation (Basile and Erijman 2008). The PheA activity was detected in all three strains (Table 2), indicating that the first intermediate product of phenol degradation by all three strains was catechol (Lee et al. 2022;Nowak et al. 2022), which is cleaved by the ortho-or meta-pathway, and the PheA activity of Ju-A4 was greater than that of the other two strains. This result corroborates previous experiments on the degradation of phenol by monocultures [ Fig. 1(b)]. Further, the degradation pathway of phenol-degrading bacteria was initially determined by detecting the enzymatic activity of C12O and C23O. The activity of C12O was detected (Table 2), while the activity of C23O was low (not shown), and it can be concluded that C23O does not dominate in this system. Among them, Pseudomonas veronii degrades catechol at low temperature following the pathway of ortho-cleavage, and this result has been experimentally demonstrated (Vogt et al. 2004). However, for Leifsonia naganoensis and Rhodococcus qingshengii, the cleavage pathways of catechol under low temperature have not been clearly reported in the literature. In the experimental system, phenol is used as the only carbon source and energy source, the pathway of ortho-cleavage to unlock the ring consumes less energy, and the meta-cleavage pathway usually occurs under medium temperature, so it is inferred that all three strains may convert the phenol degraded by the ortho-cleavage pathway.
It was detected by GC-MS that all three strains produced the intermediate product cis, cis-muconic acid. 3-hydroxyhex-2-enedioic acid was detected in Ju-A1 and Ju-A6 [ Fig. 2(c)]. The presence of oxalic acid bis(2-ethylhexyl) ester was detected by analysis of the intermediates of Ju-A4, the content of which varied with phenol [ Fig. 2(e)]. Small molecule organic acids such as oxalic acid, acetic acid and succinic acid were detected in the products of three bacterial strains. The first step in the biodegradation of phenol is dominated by the enzyme phenol hydroxylase, which  Fig. 3(a)]. Ju-A4 generates the long-chain organics in the degradation process, and these substances are not detected at the end of the reaction. This shows that the long-chain organics produced by phenol degradation can be degraded by the corresponding enzymes, and finally completely mineralized into carbon dioxide and water through the tricarboxylic acid cycle [ Fig. 3(b)].

Fitting of the response surface methodology
The response surface methodology was used to construct low-temperature phenol-degrading microbiota. Three strains were used as the three independent variables and the percentage of phenol degradation was used as the response variable. The cumulative effect of the 3 (A, B, and C) independent variables on the response variable was evaluated using a 17-set of experiments. The obtained data were subjected to regression analysis and visual image analysis.
Second order polynomial equation about response value is constructed according to three independent variables A, B and C are the three factors and the results of the quadratic analysis of variance (ANOVA) model for phenol degradation rate are given (Table 3). In this case A, B, C, A 2 , B 2 , C 2 are significant model terms, because p values greater than 0.1000 indicate the model terms are not significant. Under the interaction of three factors, the order of influence on phenol degradation rate is (expressed by coding factor): B > A > C, with Ju-A4 as the significant factor. The values of R 2 and adjusted R 2 (AdjR 2 ) are close to 1.0, which means that the regression model explains well the relationship between the factors (monoculture proportions) and the response values (removal rates). A value of "Probability > F" less than 0.05 indicates that the model term is significant (Chen et al. 2022), and a model F value of 60.69 means that the model is significant. The non-significant value of 0.3278 (greater than 0.05) for the misfit term shows that the model fits well, while the Adeq Precision of 27.892 (greater than 4) indicates that the quadratic model is statistically significant for the response values (Palla et al. 2012) and can be used for % Phenol removal =79.53 + 5.30A + 13.30B − 5.97C + 2.46AB + 0.0085AC + 0.22BC − 10.44A 2 + 5.37B 2 − 2.90C 2 .  Figure 4(a) The points in the residual normal distribution diagram are close to the straight line, indicating that it is normal distribution. The comparison diagram of residual and predicted data shows irregular distribution, which proves that the original data of all response results are stable [ Fig. 4(b)]. The predicted value and the actual value are distributed along a linear function, indicating that there is a significant correlation between the data [ Fig. 4(c)]. It is noteworthy that the predicted value of phenol degradation can be obtained in Tab. S1."

Response surface methodology analysis of maximum phenol removal rate
To investigate the interaction of the two influencing factors on the phenol removal rate, the RSM is used to plot contour plots and three-dimensional plots of the relationship between different proportional strains and phenol removal rates (Fig. 5). The design of the RSM and model fitting were described above to obtain a set of corresponding equations (expressed as coding factors) for the percentage removal of phenol. The RSM analysis of the comprehensive quadratic model shows that the optimal mixing ratio is 2:3:1 for Ju-A1: Ju-A4: Ju-A6. The validity of the model was verified at the optimal scale. The results showed that the microbiota achieved 100% phenol removal after 24 h incubation, which was consistent with the results predicted by the model (101.05%) (See Supplementary Material).

Effect of pH on the biodegradation of phenol
Changes in the pH of the reaction system affect the synthesis, secretion and catalytic activity of enzymes in microorganisms (Li et al. 2023), thus affecting the degradation of the contaminants in the system by microorganisms. The experiments investigated the degradation of phenol by the microbiota at different pH conditions, and the microbiota showed good phenol degradation ability in the pH range of 5-8, and all of them were able to complete phenol degradation within 20 h [ Fig. 6(a)]. The rate of phenol degradation was faster at pH 5. The main reason may be that the muconate lactonizing enzyme required for cis, cis-muconic acid conversion decreases in activity with increasing pH (Alva and Peyton 2003). Therefore, higher pH causes the accumulation of cis, cis-muonic acid, thus exhibiting a slower rate of phenol degradation at pH 8 than that at pH 5.

Effect of co-metabolized carbon sources on the biodegradation of phenol
The degradation of phenol is facilitated by co-metabolized substrates (Dhamale et al. 2022). Four industrially used carbon sources were selected to investigate the biodegradation of phenol in the presence of co-metabolized carbon source substrates. The experimental group with only phenol as the sole carbon source was used as the control group, and the addition of different types of carbon sources was the only variable. The complete removal of 200 mg L −1 phenol in the control group required 48 h [ Fig. 6(b)]. When the carbon source with COD equivalent of 200 mg L −1 was added, the phenol degradation rate of the experimental group with glucose and sodium citrate as co-metabolized substrates was significantly faster than the other experimental groups, and the phenol was completely degraded within 36 h. The degradation rate of the experimental group with glucose as cometabolized substrate was slightly faster than that of sodium citrate, which may be that glucose is the preferred substrate for biodegradable phenol, resulting in a faster growth rate and higher biomass yield (Shen et al. 2009). The degradation rate of the experimental group with methanol as the co-metabolized carbon source was not as good as that of the control group in the first 24 h. The reason for this is that due to the biological toxicity of methanol, the addition of methanol requires a certain adaptation period before the effect of methanol can be fully exploited, which is reflected in the degradation process after 24 h. The experimental group with the addition of sodium acetate as the co-metabolized carbon source substrate was able to degrade phenol completely within 48 h, but its degradation rate was lower than the other four groups (including the control group), probably because the low-temperature phenol-degrading strains preferentially used sodium acetate rather than phenol as the growth carbon source, and no co-metabolism system was formed between the two (Chen et al. 2021). The degradation rate of phenol became significantly faster after 36 h until complete degradation. There was a 12 h stagnation period in all experimental groups, which was not improved by the co-metabolized carbon source. Compared to the degradation without stagnation at low concentrations (i.e., concentrations less than 200 mg L −1 ), the increase in phenol concentration caused some inhibition of the microorganisms, so there was a noticeable stagnation period.

Effect of intermediate product concentration on biodegradation of phenol
Catechol is an important intermediate in the aerobic biodegradation of phenol , and the conversion of phenol to catechol by phenol hydroxylase is also a key step in determining the rate of phenol removal (Shetty and Shetty 2015), so the influence of catechol on the phenol degradation system is critical. The effect of different concentrations of catechol on the low-temperature phenol degradation system was investigated. The control group without catechol needed 48 h to completely remove phenol from the system [ Fig. 7(a)]. When 30 mg L −1 and 80 mg L −1 of catechol were added to the system, complete degradation was achieved in 36 h. The reason is that the presence of low concentrations of catechol stimulates the cells to secrete more catechol 1,2-dioxygenase to accelerate the degradation of catechol. Currently, the degradation of catechol follows the first-order degradation kinetics. It is not inhibited by the substrate concentration and is only limited by the concentration of the enzyme, so the degradation rate of catechol becomes faster and promotes the degradation of phenol. When the concentration of catechol reached 130 mg L −1 and 170 mg L −1 , the rate of phenol removal in the first 24 h was slightly inhibited compared to the control group. The reason may be due to the co-competition between the added catechol and the catechol produced by phenol metabolism, resulting in the inhibition of the active site of the enzyme. After 24 h, the phenol degradation rate was improved and gradually equalized with the control group. The reason can be analyzed from the graph of the relationship between the residual concentration of catechol in the system and the removal rate of phenol. After 24 h of degradation, the concentration of catechol in the system was greatly reduced, and the activity of catechol 1,2-dioxygenase, which affects the degradation of catechol, was higher, thus increasing the rate of phenol degradation.

Effect of substrate concentration on biodegradation of phenol and degradation kinetics
To determine the degradation ability of microbiota on phenol, the effect of different concentrations of phenol substrate on biodegradation was studied and decay kinetic parameters were calculated [ Fig. 8(a)]. It was found that the kinetic characterization of the microbiota at low concentration conditions (phenol concentrations of 100 mg L −1 and 160 mg L −1 ) proved that the phenol biodegradation in the system followed the first-order degradation kinetics at this time, and the correlation coefficients (R 2 ) were both 0.99. The biodegradation rate constants (k 1 ) and half-lives (t 1/2 ) deduced from the fitted models are shown in Table 4. The primary degradation rate constant and half-life of the microbiota were 0.16 h −1 and 4.26 h, respectively, for the degradation of 100 mg L −1 phenol. While the k 1 and t 1/2 were 0.20 h −1 and 3.52 h, respectively, when the phenol concentration was increased to 160 mg L −1 . In comparison, the primary degradation rate constant was larger and the half-life was shorter, which was caused by the full induction of catabolic genes (Tros et al. 1996). When the phenol concentration was elevated above 200 mg L −1 , the cell growth in microorganisms was closely related to the amount of substrate administered and was more consistent with the growth-related kinetic model, so a modified Gompertz model was used to calculate the relevant kinetic parameters (Table 4) (Deng et al. 2016). The modified Gompertz model described the biodegradation process of cell proliferation well with a correlation coefficient of 0.99 for all simulations. At a phenol concentration of 200 mg L −1 , the microbiota needed to adapt to a lag period of 18.81 h. The maximum biodegradation rate was 12.14 mg L −1 h −1 after acclimation. With the increase of concentration, the hysteresis time continues to increase,

Preparation and performance of low-temperature phenol-degrading microbial agent
Biological fermentation is one of the inevitable ways for microbiota to achieve industrial application in the field of pollution control. The ability of the system to resist shock loads can be increased by fermentation, and the concentration of the target compound can be maintained at a high substrate utilization rate even if it is not constant (Hughes and Cooper 1996). In this study, the low-temperature phenoldegrading microbial agent was prepared in two ways: one was to mix the monocultures separately into the fermentation medium for 48 h and then mix them according to the optimal ratio of microbiota obtained from the results of RSM experiments (fermentation first and then mixing). The other was to ferment the microbiota into the fermentation medium at a constant temperature according to the optimal ratio (mixing first and then fermentation). The effective viable bacteria count of the fermented broth was calculated by dilution coating plate method, and the effective viable bacteria count of both fermentation methods reached the order of 10 9 (CFU mL −1 ). The prepared microbial agent was compared for phenol removal performance, and the low-temperature phenol-degrading colonies constructed from unfermented strains at optimal ratios were used as the control group, with an initial OD of 0.3 for all three experimental groups. After 48-h degradation experiments, the experimental group with single strain fermentation followed by mixed preparation of microbial agent could completely degrade phenol within 36 h, and its degradation rate was enhanced by about 33% compared with the other two groups (including the control group) (Fig. 9).
The effect of single strain fermentation followed by mixed preparation of microbial agent to degrade phenol was better than that of the control group. The nature of the substrate plays a vital role in affecting microbial growth, primarily due to changes in the activity of metabolites. Microorganisms will actively respond to changes in environmental nutrients to regulate their catabolism activity (Gong et al. 2022). Most microorganisms prefer glucose for growth metabolism and do not absorb other sugars until glucose is used up (Gong et al. 2016). This is mainly due to the fact that most microorganisms have carbon catabolite repression (CCR). Since glucose is the most abundant carbohydrate monomer, efficient energy production can be achieved through oxidation of glucose. The fermentation process may be enhanced when that substrate of the ferment microorganism is maintained between the substrate restriction condition and the substrate enrichment condition (Kim et al. 2010). During fermentation, the fermenting microorganisms continuously consume glucose, thereby activating the CCR process to increase biomass. The initial COD concentration of the fermentation medium was at 5000 mg L −1 and the COD concentration at the end of the fermentation was 600 mg L −1 . A large amount of organic carbon source is consumed for microbial growth and metabolism, and the microorganisms are in a period of high growth and metabolic activity (Kim et al. 2014), and the degradation rate of phenol is higher at the same bacterial concentration.
The reason why the microbial agent prepared by fermenting first and then mixing is better than the microbial agent prepared by mixing first and then fermenting is that the initial ratio of each strain in the bacterium is destroyed due to the different growth rate of each strain during the fermentation process. Thus, it has also been demonstrated that the proportional composition of strains in the microbial agent has a direct effect on the degradation of phenol under low temperature.
Therefore, to ensure a better application effect, three strains were fermented separately and mixed according to the results of response surface methodology to construct microbial agents.

Conclusion
Three low-temperature phenol-degrading strains produced catechol during the degradation of phenol, and these intermediates followed ortho-cleavage pathway catalyzed by catechol 1,2-dioxygenase to produce cis, cis-muconic acids. The optimal ratio of the three strains to the degradation flora was obtained by RSM experiments. Phenol degradation below 160 mg L −1 followed the first-order decay kinetics, and phenol degradation in the system above 200 mg L −1 was correlated with microbial growth, and the modified Gompertz model could well predict the phenol degradation data after the stagnation period. The fermented low-temperature phenol-degrading strains were constructed in the optimal ratio of low-temperature phenol-degrading bacterial agent. The rate of phenol degradation by the agent increased by about 33% compared with the control group, and phenol could be completely degraded within 36 h. The construction of bacterial agents provides a feasible measure for the treatment of actual phenol-containing wastewater. Questions about the strain ratios of bacterial formulations and other conditions for the efficiency and pathways of treatment of phenol-containing wastewater will attract more attention from researchers.